Functional identification of DNA demethylase gene CaROS1 in pepper (Capsicum annuum L.) involved in salt stress

Pepper, which is a widely cultivated important vegetable, is sensitive to salt stress, and the continuous intensification of soil salinization has affected pepper production worldwide. However, genes confer to salt tolerance are rarely been cloned in pepper. Since the REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylase that plays a crucial regulatory role in plants in response to various abiotic stresses, including salt stress. We cloned a ROS1 gene in pepper, named CaROS1 (LOC107843637). Bioinformatic analysis showed that the CaROS1 protein contains the HhH-GPD glycosylase and RRM_DME domains. qRT-PCR analyses showed that the CaROS1 was highly expressed in young and mature fruits of pepper and rapidly induced by salt stress. Functional characterization of the CaROS1 was performed by gene silencing in pepper and overexpressing in tobacco, revealed that the CaROS1 positively regulates salt tolerance ability. More detailly, CaROS1-silenced pepper were more sensitive to salt stress, and their ROS levels, relative conductivity, and malondialdehyde content were significantly higher in leaves than those of the control plants. Besides, CaROS1-overexpressing tobacco plants were more tolerant to salt stress, with a higher relative water content, total chlorophyll content, and antioxidant enzyme activity in leaves compared to those of WT plants during salt stress. These results revealed the CaROS1 dose play a role in salt stress response, providing the theoretical basis for salt tolerance genetic engineering breeding in pepper.


Introduction
Soil salinization has been recognized as a serious environmental problem for world agriculture (Van Zelm et al., 2020;Ashraf and Munns, 2022).Salt at the bottom of the soil rises to the surface along with capillary moisture, leading to salt accumulation in the surface soil, thus causing plants to suffer from high osmotic potential.Salt stress hinders plant growth and development through ionic stress, osmotic stress, and oxidative stress (Zörb et al., 2019;Zulfiqar and Ashraf, 2021).Plants can usually adapt and maintain a balance of reactive oxygen species (ROS) under low concentrations of salt stress (Baxter et al., 2014).However, a high salt concentration leads to the excessive accumulation of ROS, which causes nucleic acid mutations and protein denaturation, leading to metabolic dysfunction and cell apoptosis (Dhindsa et al., 1981;Gill and Tuteja, 2010;Das and Roychoudhury, 2014).In order to reduce damage, plants have evolved a set of osmotic regulation, ion balance, and antioxidant defense mechanisms to enhance their salt stress tolerance (Liu et al., 2022).
5-DNA methylation is a stable and conserved epigenetic mark that occurs on the fifth carbon atom of cytosine (Vanyushin and Ashapkin, 2011) and has been implicated in genomic imprinting (Macdonald, 2012), transposable element repression (Xie et al., 2013), X-chromosome inactivation (Bala Tannan et al., 2014), gene silencing (Fu et al., 2013), and other growth and development processes.DNA methylation is usually present in symmetric CG sequences in mammals, but also occurs in asymmetric CHH sequences (where H is A, C, or T) and symmetrical CG or CHG sequences in plants (Law and Jacobsen, 2010;He et al., 2011).These modifications are mainly carried out by methyltransferases, chromomethyltransferases, and domain-rearranged methyltransferases (Liu et al., 2018).DNA methylation can be triggered by environmental changes, which prompts the opposite functions of DNA demethylase and methyltransferase to regulate DNA methylation levels (Cao et al., 2014).
Under abiotic stress, DNA methylation and demethylation work together to regulate DNA methylation levels of key genes (oxidoreductase and regulating ion balance), resulting in changes in gene expression, so as to regulate plant tolerance to stress at the physiological level (Bharti et al., 2015;Ferreira et al., 2015).REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylase whose demethylation activity is involved in base excision DNA repair and protects the genome from harmful methylation during plant development (Gong et al., 2002;Morales-Ruiz et al., 2006;Parrilla-Doblas et al., 2019;Khouider et al., 2021).Studies have shown that the products of the ROS1 gene contribute to the regulation of plant flowering and fruit development (Lang and Gong, 2018;Liu et al., 2018).Moreover, ROS1 gene is closely associated with the plant response to abiotic stresses.For example, the DNA methylation in the promoter region of genes associated with antioxidant pathways is reduced under salt stress in tobacco plants overexpressing AtROS1, leading to an increase in gene expression levels of antioxidant enzymes (such as glutathione S-transferase, ascorbate peroxidase, glutathione peroxidase and glutathione reductase) and enhanced ROS removal capacity, thereby enhancing salt tolerance (Bharti et al., 2015).Gahlaut et al. found differences in the expression of wheat dMTase proteins (ROS1 and DEMETER-Like) in different developmental stages, tissues, and varieties under heat stress (Gahlaut et al., 2022).The expression of the DNA demethylase HcROS1 was increased, the level of genomic DNA methylation was decreased, and the plants' salt tolerance was enhanced (Du et al., 2017).Under cold stress, Arabidopsis ROS1 mediated DNA demethylation plays important roles in the transcription of defense genes and stress response genes (Yang et al., 2022).Sun et al. found that ZmROS1 helps to regulate methylation in maize under salt stress (Sun et al., 2017).The overexpression of the products of the gene NtROS2a in tobacco could increase plants' tolerance to abiotic stress, similar to the Arabidopsis ROS1 gene (In Hye et al., 2015).
Pepper (Capsicum annuum L.) is a Solanaceous vegetable originating from Central and South America, and it is an industrial raw material for medicine, spicy condiments, and cosmetics (Zou and Zhu, 2022).China is the country that produces and consumes the most chili peppers in the world (Zhang et al., 2023).Salt stress is one of the important reasons for pepper yield loss and it adversely affects pepper growth and development.Although the function of the demethylase ROS1 has been investigated in many species, it has not been isolated and its function remain unclear in pepper.Therefore, we characterized the biological effects of CaROS1 genes on the response to salt stress in pepper by gene silencing and overexpressing technologies.These results help us to understand the function of CaROS1 and provide a theoretical basis for cultivating salt-tolerant varieties in pepper 2 Results

Identification and bioinformatics analysis of CaROS1
CaROS1 (LOC107843637), which consists of 19 exons and 18 introns, is located on chromosome 10 of pepper genome (Figure 1A).The length of CaROS1 coding sequence is 5,451 bp, and the CaROS1 gene encoding a 201.16 kDa protein containing 1,816 amino acids, with a PI of 6.92.The instability index of CaROS1 is 49.17, which means that it is considered an unstable protein (Figure 1B).The maximum hydropathicity value of CaROS1 is 1.83, and the minimum is -3.79 (Supplementary Figure S1A), thus, it is a hydrophilic protein.The CaROS1 protein has neither an obvious signal peptide nor a transmembrane domain (Supplementary Figures S1B, C).The secondary structure of CaROS1 comprises alpha helices (30.40%), beta turns (2.92%), extended strands (9.97%), and random coils (56.72%) (Figure 1C).Based on the secondary structure, the tertiary structure of CaROS1 is shown in Supplementary Figure S1D.

Phylogeny and domain analysis of ROS1
The alignment of ROS1 proteins between pepper and other species using MEGA-X software showed that the ROS1 proteins in Solanaceous plants (such as tomato, potato, wolfberry and tobacco) have the closer evolutionary relationships to those of in pepper (Figure 2A).However, the evolutionary relationships between the ROS1 proteins of Solanaceae and Cruciferae plants is relatively distant.Then, a multiple sequence alignment of the ROS1 proteins among Solanaceae and Cruciferae plants, including Arabidopsis, flax, Eutrema and white mustard, was performed (Figure 2B).The results show that all ROS1 proteins contain a highly conserved HhH-GPD glycosylase domain and a RRM_DME domain.The HhH-GPD domain has a helix-hairpin-helix (HhH) motif, a Gly/ Pro rich loop followed by a conserved aspartate (GPD), and a C terminal 4Fe-4S cluster.The RRM_DME structure is a predicted folded domain of an RRM-like glycolyase, which is important for RNA regulation and promoting the interaction of catalytic domain with ssDNA.

Subcellular localization and expression patterns of CaROS1
To examine the subcellular localization of CaROS1, the 35S:: CaROS1-GFP construct was transiently expressed in tobacco leaves.The fluorescence signal of CaROS1 was detected in both the nucleus and cytoplasm of tobacco epidermal cells.This is consistent with the fluorescence signal detected for the control GFP (Figure 3A).
To investigate the expression patterns of CaROS1, its relative expression levels in different tissues, including roots, stems, leaves, Evolutionary tree and multiple sequence alignment of ROS1 proteins.flowers, young fruits, and mature fruits, were calculate based on qRT-PCR.As shown in Figure 3B, CaROS1 was highly expressed in young fruits, followed by mature fruits, leaves, and roots, with the lowest expression in stems.In addition, after treated with different NaCl concentrations (0 mM, 50 mM, 100 mM, and 200 mM), we found that the expression of CaROS1 was significantly increased in the 100 mM and 200 mM NaCl treatment groups (Figure 3C).

Influence of CaROS1 silencing on salt tolerance in pepper
The expression of CaROS1 was induced by salt stress (Figure 3C), indicating that CaROS1 was involved in salt stress response regulation.Therefore, virus-induced gene silencing (VIGS) was used to explore the effect of CaROS1 silencing on salt tolerance in pepper.After 25 days of transient transformation, leaves of TRV: 00 (control plants) and TRV: CaROS1 plants were collected, and the CaROS1 gene expression levels were determined using qRT-PCR.The CaROS1 gene expression in gene silenced plants was 77.3% lower than that in the WT plants (Supplementary Figure S2).
The phenotypes of TRV: 00 and TRV: CaROS1 were not significantly different under normal conditions, but a curling tip was found in parts of the control plants and CaROS1-silenced plants under 50 mM NaCl treatment.After the 100 mM NaCl treatment, the leaves of TRV: CaROS1 plants were slightly withered, while only parts of the TRV: 00 plants were curled.The CaROS1 gene silenced plants showed obvious symptoms of wilt and sagging, and the degree of damage was significantly greater than that observed for the control plants under the 200 mM NaCl treatment (Figure 4A).
Nitroblue tetrazolium (NBT) staining was used to detect the accumulation of H 2 O 2 and O 2-in the plants.The leaves of the silenced plants showed more blue-stained spots than those of the control plants under NaCl stress (Figure 4B).After treatment with 100 mM and 200 mM NaCl, the relative electrolyte content (REC) and malondialdehyde (MDA) content of TRV: CaROS1 plants were significantly higher than TRV: 00 plants (Figures 5A, C).Compared to that in the TRV: 00 plants, the content of proline (Pro) in the TRV: CaROS1 plants was significantly increased (Figure 5B).
Salt stress can induce ROS production, so it is important to explore the activity of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) in removing excess ROS.The POD and CAT activities in CaROS1-silenced plants were significantly higher than those in the TRV:00 plants after the 100 mM NaCl treatment, but significantly lower than those in the control plants after the 200 mM NaCl treatment (Figures 5D, E).Furthermore, the SOD activity of TRV: CaROS1 plants was significantly lower than that in the control plants under NaCl treatment (Figure 5F).

Resistance of transgenic CaROS1 tobacco seedlings to salt
To further validate the mechanism underlying the CaROS1 response to salt stress, we produced 'k326' tobacco plants with CaROS1 gene overexpression (Supplementary Figure S3).After analyze the expression of the CaROS1 in different transgenic tobacco lines, the overexpression-4 (OE-4) and OE-7 lines were selected for subsequent experiments because of their high CaROS1 expression (Supplementary Figure S4B).Twenty-day-old wild-type (WT) and OE lines were treated with 0, 50, 100, and 200 mM NaCl salt for 22 days.There was no significant phenotypic difference between the OE lines and the WT plants under normal conditions (Figure 6A).However, the leaves of the WT plants showed obvious chlorosis and yellowing, while the growth of the OE lines was less affected by salt stress.Moreover, the Na + and relative electrolyte contents of the WT plants were significantly increased compared to those of the OE lines under the 100 and 200 mM NaCl treatments (Figures 6B, D).In contrast, the relative water content (RWC) and total chlorophyll content in the WT plants were significantly decreased compared to those in the OE lines (Figures 6C, E).
There was no significant difference in SOD and POD activity between the WT plants and OE plants under normal and low-salt (50 mM NaCl) conditions.After the 200 mM NaCl treatment, the SOD and POD activities of the OE lines were significantly increased compared to those of the WT plants.These results indicate that the increased salt tolerance is related to the overexpression of CaROS1 in transgenic tobacco.
Subcellular localization helps us to understand the function of genes.It was found that ROS5 in Arabidopsis is located in nucleus, functions as a nuclear transporter, and is required to prevent gene hypermethylation (Zhao et al., 2014).Gong et al (Gong et al., 2002).observed AtROS1 is located in nucleus, and it has regulatory effects Effect of CaROS1 overexpression on salinity stress tolerance in tobacco.on gene expression and epigenetics triggered by smRNAs.We revealed that CaROS1 is located in the cytoplasm and nucleus (Figure 3A).This implies that CaROS1 not only plays a role in regulating transcription, but also associated with the function of cytoplasmic transporters.This result also suggests that the ROS1 homologs in different species may show inconsistent subcellular localization.Previous studies have investigated the expression characteristics of ROS1 in different tissues.Liu et al (In Hye et al., 2015).reported that the OsROS1 in rice is highly expressed in mature pollen, anthers, and the ovary, but rarely expressed in roots and endosperm.Xu et al (Xu, 2023).reported that ZmROS1a and ZmROS1b in maize are highly expressed in embryo and endosperm.In this study, we found CaROS1 is highly expressed in young fruits of pepper, followed by mature fruits and flowers, and has the lowest expression in the stem (Figure 3B).These results suggest that CaROS1 gene may be involved in the regulation of floral organ development and fruit ripening.Furthermore, CaROS1 gene expression increased with increasing NaCl concentration under salt stress (Figure 3C).This result suggests that there is a positive relationship between the expression of CaROS1 and the salt stress dose response in pepper seedlings.
VIGS is a gene transcriptional repression technique used to study gene function in plants.It is used as a reverse genetic tool for the post-transcriptional silencing of a specific gene before phenotypic identification and gene expression in order to determine the roles of target genes in plant growth and development (Ramegowda et al., 2014).The CaROS1 gene was silenced in pepper using VIGS to explore the role of this gene in the salt stress response.The results showed that the TRV: CaROS1 plants were more sensitive to salt stress than the TRV: 00 plants (Figure 4A).ROS include hydrogen peroxide (H 2 O 2 ), superoxide (O 2-), and hydroxyl radical (OH -), which are byproducts of aerobic metabolism (Mansour and Salama, 2019).Under salt stress, a high accumulation of ROS causes the degeneration of cellular proteins, leading to enzyme inactivation, nucleic acid degradation, cell membrane damage, carbohydrate oxidation, and ultimately cell death (Mansour and Salama, 2020;Mansour and Hassan, 2022).In our study, we found that the proline content of CaROS1-silenced plants was lower than that of the control plants under salt stress, while the MDA content showed the opposite trend (Figures 5B, C).There is an adaptive water retention mechanism in organisms under stress that is related to the de novo synthesis of compatible solutes (glycine betaine, proline, sugars, and polyamines) and ion flux across cellular membranes (Hameed et al., 2021).Under abiotic stress, these substances can make cells more tolerant, and proline is known to protect the photosynthetic apparatus in cytokinindependent photorespiration under salinity.Strong exogenous stress can cause an increase in proline.Thus, the severe damage in CaROS1-silenced plants caused by salt stress, making them unable to accumulate the large amounts of proline needed to maintain the cell's osmotic potential (Ashraf and Foolad, 2007;Shin et al., 2020).In contrast, When CaROS1-silenced subjected to salt stress, superoxide free radicals are produced for the peroxidation of membrane lipids to produce a large amount of malondialdehyde.The excessive accumulation of MDA causes the cross-linking polymerization of vital macromolecules such as proteins and nucleic acids, leading to changes in the structure and function of the cell membrane (Guo et al., 2019).The accumulation of ROS induced by environmental stress causes plants to eliminate ROS through the signal transduction of the synthetic antioxidant enzyme system, which includes superoxide dismutase SOD, CAT and POD (Acet and Kadıoglu, 2020;Wang et al., 2021).After the 50 mM and 100 mM NaCl treatments, the POD and CAT activities of TRV: CaROS1 plants were increased compared to those of the TRV: 00 plants (Figures 5D, E).This is because CaROS1 silenced plants require higher CAT and POD enzyme activity to alleviate the damage caused by low concentration of salt stress.In contrast, under 200 mM NaCl stress, the TRV: CaROS1 plants had a lower antioxidant enzyme activity than the TRV: 00 plants (Figures 5D, F).These results indicate that, when salt concentrations are too high, CaROS1-silenced plants fail to remove large amounts of ROS, causing severe damage to the plants, indicating that the CaROS1 gene may be critical for the survival of pepper plants in heavily salt polluted environments.
CaROS1 gene was then overexpressed in tobacco and the transgenic plants were treated with different NaCl concentrations to further confirm the role of CaROS1 gene in salt stress.WT plants showed more severe damage than the OE lines under salt stress.Under high salt stress, the leaves of control plants suffered severely chlorosis, and some leaves turned yellow and even withered, while the OE lines grew well (Figure 6A).This result is consistent with previous studies of ROS1 under salt stress (Bharti et al., 2015).In addition, with increasing salt concentrations, the chlorophyll content decreased, while the accumulation of Na + increased (Figures 6B, E).The toxicity of salt stress mainly arises from ion toxicity and osmotic stress (Zhang et al., 2020;Liu et al., 2022).Under salt stress, the content of Na + and Cl -increased in plants, which reduced the affinity between chlorophyll and chlorophyll proteins, and finally chlorophyll dissociated (Loudari et al., 2020).Moreover, severe salt stress will inhibit the activity of key enzymes in chlorophyll synthesis, activate chlorophyll enzymes and accelerate their degradation (Athar et al., 2022).When the salt concentration of soil increased, the soil's water potential is lower than that of the plant's root tip cells, which prevents water absorption through the plant's roots, leading to a water shortage and osmotic stress in the plant (Lv, 2014).In this study, the relative water content in OE lines did not change significantly, however, in control plants it decreased with increasing salt treatment concentrations (Figure 6C).Furthermore, salt stress disrupts the balance between ROS production and clearance in plant cells, resulting in increasing ROS concentrations that activate ROSscavenging enzymes.After 200 mM sodium chloride treatment, we found the CaROS1 overexpressing plants had higher SOD and POD activity than WT plants (Figures 6F, G).The above results indicate that transgenic tobacco overexpressing CaROS1 showed tolerance to salt stress.This is similar to previous studies on overexpression of AtROS1 under abbiotic stress (Bharti et al., 2015;Yang et al., 2022;Bharti et al., 2023).
To summarize, we isolated the CaROS1 gene, revealed its expression patterns, analyzed the function of CaROS1 by gene silencing and overexpressing strategy, and investigated its possible physiological role.Overall, CaROS1 was found to positively regulate the salt stress tolerance in pepper.

Experimental materials and treatments
In this study, the pepper variety "zunla-1" was provided by the Vegetable Research Institute of the Guangdong Academy of Agricultural Sciences, and the "k326" tobacco was provided by the College of Horticulture and Landscape Architecture (Zhongkai College of Agricultural Engineering).Pepper seeds were disinfected with 70% ethanol and 1% sodium hypochlorite and then washed with ultrapure water and soaked for 12 h, and sprouting was accelerated in a 28 °C incubator culture.Germinated seeds were selected and sown into vegetative soil and grown in an incubator at 25 °C in light (16 h) and 20 °C in darkness (8 h) until 4-5 true leaves appeared.For the CaROS1 tissue-specific expression analysis, six tissues, including root (R), stem (S), leaf (L), flower (F), young fruit (YF), and mature fruit (MF), were collected.For the gene-silenced pepper, samples were collected from TRV: CaROS1 and TRV: 00 plants after 20 days of NaCl stress at 0, 50, 10, and 200 mM.

Bioinformatics analysis of CaROS1
The physicochemical properties of CaROS1 proteins, including their amino acids, isoelectric points (PI), instability indices, and protein weights, were analyzed using the National Center for Biotechnology Information and ExPASy platforms.Gene structure maps were drawn using GSDS.The pro/hydrophobic regions of the CaROS1 protein were analyzed using ProtScale, and the signal peptide and transmembrane domains were predicted using SignaIP 4.1 and TMHMM 2.0.The secondary and tertiary structures of the CaROS1 proteins were analyzed using NPSA-PRABI and SWISS-MODEL.Links to the bioinformatics analysis website are provided in Supplementary Table S1.

Expression analysis of CaROS1 gene
Total RNA was isolated from pepper and tobacco samples using the Total RNA Kit I (OMEGA, Guangzhou, China), and the concentrations were detected using an ND-100C ultrascopic UV visible spectrophotometer (MIULAB, China).The first strand cDNA was synthesized with the Hifair® 1st Strand cDNA Synthesis Super Mix for qPCR kit (Yeasen, Shanghai, China).The qRT-PCR was performed using the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Shanghai, China) and 2×ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China).The relative expression of CaROS1 was analyzed using the 2 -DDCt method (Livak and Schmittgen, 2001).The primer information for CaROS1 is provided in Supplementary Table S2.

Subcellular localization of CaROS1
In order to construct the 35S::CaROS1-GFP fusion protein, the coding sequence of CaROS1 was cloned into the N terminus of GFP under the control of the CaMV-35S promoter in the vector pNC-Cam1304-SubN (Chinese Academy of Tropical Agricultural Sciences, China).The 35S::CaROS1-GFP constructs were then transformed into tobacco leaves (Zhao et al., 2017).Fluorescent signals were observed and images were taken using an FV1000 confocal laser microscope (OLYMPUS, Japan).For the GFP protein and nuclear marker location observation, 488 nm and 561 nm excitation spectrum, 510 nm and 580 nm emission spectrum, was used in this experiment, respectively.

VIGS of pepper seedlings
The coding region of CaROS1 was joined to the pNC-TRV2 vector (Chinese Academy of Tropical Agricultural Sciences) to form pTRV2:CaROS1.Then, pTRV1 (Chinese Academy of Tropical Agricultural Sciences), pTRV2:00 (control), and pTRV2:CaROS1 were transformed into Agrobacterium tumefaciens strain GV3101 (WeGo, Guangzhou, China).CaROS1 was silenced in pepper using the method described by Singh et al (Singh et al., 2022).After 25 days of silencing, leaves of TRV2:CaROS1 plants and pTRV2:00 plants were collected to measure the silencing efficiency.

Construction of overexpressed CaROS1 cloning vector
The CaROS1 sequence was amplified by PCR using the 2×Hieff Canace® Plus PCR Master Mix (Yeasen, Shanghai, China).The purified amplified products were ligated into the pNC-Cam1304-35S vector (Chinese Academy of Tropical Agricultural Sciences) and transfected into Trans1-T1 Phage Resistant chemocompetent cells (WeGo, Guangzhou, China).Then, they were placed on Luria-Bertani (LB) (50 mg/mL Kan) solid medium for 12 h, and single E. coli colonies were expanded in LB liquid medium (37 °C, 210 rpm) to an OD 600 of 0.65.The positive clones were identified by PCR and sent to Sangon Biological Company (Shanghai, China) for sequencing confirmation.

Tobacco conversion
The "k326" tobacco seeds were sterilized and sown into MS (Murashige Skoog) medium until 3-4 true leaves grew.CaROS1 transgenic tobacco was obtained using Agrobacterium tumefaciensmediated leaf disc transformation (Oh et al., 2005).In addition, the lines were identified using qRT-PCR during the T1 seedling period (Supplementary Figure S4).T1 tobacco seeds were further screened for T2 lines on MS medium containing 50 mg/mL kanamycin.The T3 line was used for the subsequent experiments.

Salt-stress treatment and sample collection
Pepper seeds were soaked for 8 h, germinated in a 28 °C incubator, and seeded into vegetative soil (matrix, vermiculite, and perlite mixed at 3:1:1 ratio).Each plant was planted in an independent basin (same basins were used).Five-six true leaf stage pepper seedlings were subjected to salt stress for 0, 50, 100 and 200 mM.The 50 ml of NaCl solution was poured into the soil of each pepper plant for every four days.Samples were collected after 20 days of salt stress treatment.For the treatment of silenced plants, TRV: CaROS1 and TRV: 00 plants were treated with 0, 50, 100 and 200 mM NaCl solution for 20 days.The pepper leaves were then harvested and all samples were immediately placed in liquid nitrogen and then stored at-80 °C.
"K326" tobacco was used for gene transformation in this experiment.Seeds of wild-type tobacco and transgenic lines were soaked in water, vernalized at 4 °C for 48 h, and then sown in a high-pressure-sterilized nutrient mixture.The tobacco was placed in a temperature incubator (26 °C light (16 h)/20 °C dark (8 h) conditions) for 20 days.Then they were placed in a big pot and watered every four days with different concentrations of NaCl solution (500 ml per basin) at room temperature.Samples were collected after 22 days of salt stress treatment.

NBT staining
We performed NBT staining of pepper leaves following Wohlgemuth's method (Wohlgemuth et al., 2002).TRV2:00 and CaROS1-silenced pepper leaves were immersed in NBT staining solution in the dark for 12 h, then added to 50 mL of absolute ethanol and heated to decoloring at 100 °C.The concentration of the NBT staining solution was 0.5 mg/mL.

Determination of plant physiological indicators
We measured the REC and RWC as described by Choluj et al (Chołuj et al., 2014). andPan et al (Pan et al., 2012), respectively.The MDA content, proline content, and activity of antioxidant enzymes (POD, SOD, and CAT) were measured using a plant detection kit (Nanjing Jiancheng Biotech, China), according to the manufacturer's instructions.The Na + content and total chlorophyll content were measured as described by Li and Arkus (Arkus et al., 2005;Li, 2015).

Statistical analysis
Statistical analysis was performed using SPSS Statistics 26.0.Differences were considered significant at P < 0.05 and extremely significant at P < 0.01.

Conclusions
We cloned the CaROS1 gene, which encodes a protein that contains the highly conserved HhH-GPD and RRM _ DME domains and localizes in the nucleus.According to the qRT-PCR analysis, CaROS1 was highly expressed in the young and mature fruit of pepper, and was rapidly induced by salt stress.We verified its biological function through the silencing and overexpression of CaROS1 in pepper and tobacco, respectively.Compared with control plants, CaROS1-silenced plants showed more severe damage, lower antioxidant enzyme activity, and reduced tolerance to salt stress.The CaROS1 transgenic tobacco showed higher relative water content and total chlorophyll content than the WT plants, and ROS accumulated less in the transgenic tobacco than in the WT plants.The above results indicate that CaROS1 gene is valuable in regulating the salt tolerance of pepper.
FIGURE 1 Basic information for CaROS1 in pepper.(A) Structure of CaROS1 gene.Red rectangles and black lines indicate exons and introns, respectively; blue rectangles indicate the upstream and downstream sequence of CaROS1.(B) Physicochemical properties of CaROS1.(C) The predicted secondary structure of CaROS1.
FIGURE 3 Subcellular localization and expression analysis of CaROS1.(A) The 35S::GFP and 35S::CaROS1 plasmids were separately transformed into tobacco leaves for subcellular localization analysis.GFP is shown in green, and nuclear markers in red.Scale bar = 20 mm.(B) Analysis of the expression patterns of CaROS1 in pepper; root (R), stem (S), leaf (L), flower (F), young fruit (YF), and mature fruit (MF).The expression level of CaROS1 in the root was set to 1. (C) Leaves were treated with different concentrations of NaCl (0, 50, 100, and 200 mM) for 20 days, and then the expression pattern of CaROS1 under salt stress was determined using qRT-PCR."**" indicates P < 0.01.